Alterations in the Gut Fungal Community in a Mouse Model of Huntington’s Disease

ABSTRACT Huntington’s disease (HD) is a neurodegenerative disorder caused by a trinucleotide expansion in the HTT gene, which is expressed throughout the brain and body, including the gut epithelium and enteric nervous system. Afflicted individuals suffer from progressive impairments in motor, psychiatric, and cognitive faculties, as well as peripheral deficits, including the alteration of the gut microbiome. However, studies characterizing the gut microbiome in HD have focused entirely on the bacterial component, while the fungal community (mycobiome) has been overlooked. The gut mycobiome has gained recognition for its role in host homeostasis and maintenance of the gut epithelial barrier. We aimed to characterize the gut mycobiome profile in HD using fecal samples collected from the R6/1 transgenic mouse model (and wild-type littermate controls) from 4 to 12 weeks of age, corresponding to presymptomatic through to early disease stages. Shotgun sequencing was performed on fecal DNA samples, followed by metagenomic analyses. The HD gut mycobiome beta diversity was significantly different from that of wild-type littermates at 12 weeks of age, while no genotype differences were observed at the earlier time points. Similarly, greater alpha diversity was observed in the HD mice by 12 weeks of age. Key taxa, including Malassezia restricta, Yarrowia lipolytica, and Aspergillus species, were identified as having a negative association with HD. Furthermore, integration of the bacterial and fungal data sets at 12 weeks of age identified negative correlations between the HD-associated fungal species and Lactobacillus reuteri. These findings provide new insights into gut microbiome alterations in HD and may help identify novel therapeutic targets. IMPORTANCE Huntington’s disease (HD) is a fatal neurodegenerative disorder affecting both the mind and body. We have recently discovered that gut bacteria are disrupted in HD. The present study provides the first evidence of an altered gut fungal community (mycobiome) in HD. The genomes of many thousands of gut microbes were sequenced and used to assess “metagenomics” in particular the different types of fungal species in the HD versus control gut, in a mouse model. At an early disease stage, before the onset of symptoms, the overall gut mycobiome structure (array of fungi) in HD mice was distinct from that of their wild-type littermates. Alterations of multiple key fungi species were identified as being associated with the onset of disease symptoms, some of which showed strong correlations with the gut bacterial community. This study highlights the potential role of gut fungi in HD and may facilitate the development of novel therapeutic approaches.

during adulthood. What is the relationship between age, gut inflammation, motor dysfunction and alterations in the mycobiome?
Reviewer #2 (Public repository details (Required)): The Microbiome raw sequencing data should be submitted and the link should be clearly findable in the paper.

Reviewer #2 (Comments for the Author):
This is a manuscript on the role of the bacteriome and mycobiome in a mouse model of huntington's disease. The authors find some alterations in the micorbiome and mycobiome in the mice with HD, which is unsurprising given the genetic nature of the disease. Generally, the text is well-written. The authors use long bold headers for their paragraphs, which I found helpful to quickly pick up the message. The bioinformatics analysis is reasonable and the conclusions follow from the results. Generally, the manuscipt is in good shape. I have written down some comments below.
Major comments: Huntington's desease is a relatively well-understood disease with a clear genetic origin. Because of this, it would be very interesting to take this analysis to a functional level ans see if any genes in the microbiome that are in any way related to Huntington's disease are altered in the bacteriome and/or mycobiome.
It is not clear why the authors used a Kruskal Wallis test to determine an increase in HD mice at week 12. I see the argument for using a non-parametric test as variance is unequal over time, but KW is typically used for more than two groups. If in fact the KW was ran over all 10 groups, a post-hoc is necessary to determine differences between two groups.
Similarly, for the PERMANOVA in fig1C, is this a test for the entirity of the data or just for the HD vs WT of that age group?
It is likely a post-hoc correction as well as post-hoc test would be warranted in both cases.

Minor comments:
The authors analyse longitudinal micorbiome data and briefly investigate the temporal stability of several taxa. Change over time in the microbiome is known as volatilty and in their previous work (10.1016/j.nbd.2020.105199), the authors do indeed measure bacteriome volatility in the same animals (though they did not refer to the original manuscript describing volatility in either of their manuscripts: 10.1016/j.psyneuen.2020.105047). Why do the authors not perform the same analysis here?
The authors use the term dysbiosis. This term has lost favour in the field as it is hard to define. (A microbiome does not have to be unhealthy for it to make the host sick) Please rephrase.

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Response to Reviewers
We thank the Editor for obtaining constructive peer review and the Reviewers for taking the time to share their expertise and help us improve our manuscript. We have extensively revised our manuscript to address each and every comment from the Reviewers. We detail our responses below, and in the revised manuscript. Many thanks for your kind assistance and consideration.

Reviewer #1
In this study, the authors provides evidence of an altered gut fungal community (mycobiome) in the R6/1 transgenic mouse model of HD. This a very-well written study that provides novel data to the field.

Although the authors used a relatively small number of animals (n=9 /genotype), the longitudinal collection of fecal samples from 4-to 12-weeks is very interesting, as it
offers the opportunity to correlate individual mouse "disease trajectory" with changes in the mycobiome. However, no attempt was made to correlate any behavioral abnormalities with changes in the mycobiome (e.g., alpha diversity)?
RESPONSE: As behavioural tests have been shown to affect gut microbiome composition, we kept this cohort of mice as behaviourally naïve as possible aside from rotarod testing, which assesses motor coordination. We did not correlate this with changes in mycobiome alpha diversity, as alpha diversity is challenging to interpret since both increased and decreased alpha diversity has been associated with negative health outcomes. However, we did integrate the longitudinal mycobiome dataset with longitudinal rotarod data. Since many of mice did not show any significant impairments in rotarod performance by Week 12, the integration resulted in minimal correlations and very poor performance of the machine learning model. Similar results were obtained when integrating body weight data with mycobiome composition. Thus, we did not include these results.
The authors should modify the title and clarify "in a mouse model of Huntington's disease".

RESPONSE: The title has been amended.
The authors should include other information about the animals (e.g., sex, presumably males?, weight at each of the 5 points).
RESPONSE: Yes, male mice were used in the study. The weight of each mouse at the 5 time points and motor performance based on rotarod have been included in the Supplementary Data.
What is driving the changes at 12 weeks of age? Previous studies have shown gut inflammation in the same mouse model during adulthood. What is the relationship between age, gut inflammation, motor dysfunction and alterations in the mycobiome? RESPONSE: We agree it is a very important question. Huntington's disease is caused by a genetic mutation in the huntingtin (HTT) gene which is also expression in the gut. We believe that the changes in the gut microbiome is primarily due to expression of the genetic mutation, which could alter gut structure, function and induce gut inflammation, resulting in gut microbiome alteration. Given that Huntington's disease is a progressive disease, it is unknown exactly at what age does gut inflammation and significant damage to the gut architectural structure occur. However, this is beyond the scope of this study as the aim of is to examine the gut mycobiome composition and its relation to the gut bacteriome.

Reviewer #2
The Microbiome raw sequencing data should be submitted and the link should be clearly findable in the paper. RESPONSE: As this is an analysis of an already published dataset, the raw sequencing data is available on the NCBI portal and can be searched using the BioProject Number PRJNA613182 provided in the Methods section (Line 320). This is a manuscript on the role of the bacteriome and mycobiome in a mouse model of huntington's disease. The authors find some alterations in the micorbiome and mycobiome in the mice with HD, which is unsurprising given the genetic nature of the disease. Generally, the text is well-written. The authors use long bold headers for their paragraphs, which I found helpful to quickly pick up the message. The bioinformatics analysis is reasonable and the conclusions follow from the results. Generally, the manuscript is in good shape. I have written down some comments below.
Major comments: Huntington's disease is a relatively well-understood disease with a clear genetic origin. Because of this, it would be very interesting to take this analysis to a functional level and see if any genes in the microbiome that are in any way related to Huntington's disease are altered in the bacteriome and/or mycobiome.

RESPONSE:
The functional analysis of this shotgun sequencing data and its relation to the changes in HD gut bacteriome has been investigated and published previously (10.1016/j.nbd.2020.105199). We did not apply the same analysis here because the fungal database (mycobiome) is more sparsely annotated compared to its bacteriome counterpart and there are many unculturable fungi yet to be discovered with various unknown function and taxonomy. Additionally, fungi are often mislabelled in databases which complicates taxonomy assignment. Thus, additional functional relationships to the mycobiome will not be reliable, and could be misleading.